Zusammenfassung
The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds
nonribosomally via the thiotemplate mechanism. Acyladenylation,
thioesterification, N-methylation, and cyclization of two amino
acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase.
Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces
acidiscabies 84.104 identified an open reading frame (ORF) encoding
a homolog of the P450 monooxygenase gene family. It was proposed
that thaxtomin A phenylalanyl hydroxylation was catalyzed by the
monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104
by using an integrative gene disruption construct, and culture filtrate
extracts of the mutant were assayed for the presence of dehydroxy
derivatives of thaxtomin A. Reversed-phase high-performance liquid
chromatography (HPLC) and HPLC-mass spectrometry indicated that
the major component in culture filtrate extracts of the mutant was
less polar and smaller than thaxtomin A. Comparisons of electrospray
mass spectra as well as (1)H- and (13)C-nuclear magnetic resonance
spectra of the purified compound with those previously reported
for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl),
the didehydroxy analog of thaxtomin A. The ORF, designated txtC,
was cloned and the recombinant six-His-tagged fusion protein produced
in Escherichia coli and purified from cell extracts. TxtC produced
in E. coli exhibited spectral properties similar to those of cytochrome
P450-type hemoproteins that have undergone conversion to the catalytically
inactive P420 form. Based on these properties and the high similarity
of TxtC to other well-characterized P450 enzymes, we conclude that
txtC encodes a cytochrome P450-type monooxygenase required for postcyclization
hydroxylation of the cyclic dipeptide.
Nutzer